The present invention relates to cell storage and delivery systems. More specifically, the present invention is directed to biodegradable and/or bioabsorbable fibrous articles having utility as a carrier for viable cells.
Methods and compositions for encapsulating core materials, e.g., materials containing drugs, have been disclosed. These methods generally involve encapsulating the core material within a microcapsule by forming a semipermeable membrane around the core material.
Although a number of processes for microencapsulation of core material have been developed, most of these processes cannot be used for pH, temperature or ionic strength-sensitive material such as viable cells because of the harsh conditions necessary for encapsulation. U.S. Pat. No. 4,352,883 to Lim discloses what is believed to be one of the first processes for successfully encapsulating viable tissue or cells within a semipermeable membrane. In the process, a temporary capsule of a gellable material, e.g., an anionic gum such as sodium alginate, is formed about the tissue or cells and a permanent, semipermeable membrane is formed by cross-linking surface layers of the temporary capsule. Specifically, a mixture of the gum and the core material is subjected to a gelling solution, preferably a calcium ion solution, to produce a temporary capsule. The resulting temporary capsule is reacted with a solution of a polycationic material to form a permanent membrane. The interior of the capsule may be reliquified by reestablishing conditions under which the anionic gum is liquid, e.g., changing the ionic environment by placing the capsules in phosphate buffered saline. Reliquification of the interior of the capsule facilitates nutrient transport across the membrane, promoting cell growth. The process need not damage the core material or hamper the viability of cells because the temperature, ionic strength, and pH ranges used in the encapsulation process need not be harsh.
A preferred embodiment of the Lim encapsulation technique involves the formation of shape-retaining gelled masses that contain the material to be encapsulated, followed by deposition of a membrane on the surface of the gelled masses. The membrane is formed as relatively high molecular weight materials contact the gel masses and form ionic cross-links with the gel. Lim discloses that lower molecular weight cross-linking polymers permeate further into the structure of the gelled masses and result in a reduction of pore size. Lim also discloses that the duration of membrane formation affects pore size. Given a pair of reactants, the longer the cross-linking polymer solution is exposed to the gelled mass, the thicker and less permeable the membrane.
While the techniques for porosity control and membrane formation disclosed in the Lim patent may form acceptable membranes, they do not allow fine tuning of the membrane porosity, but rather set rough differential filtering limits.
In addition to improved porosity, for commercial purposes it is also important to be able to consistently produce microcapsules in large numbers having defect-free membranes. In this regard, membranes formed by the Lim techniques occasionally have protruding portions of cells or have cells anchored on the capsules. The Lim techniques also may produce capsules containing voids that allow cells, the substance of interest, or unwanted contaminants to escape from the capsule. If a small fraction of the microcapsules made with a specific purpose in mind have membrane voids, many of the objectives and advantages of the processes would be frustrated. Accordingly, encapsulation processes that promote membrane uniformity and avoid random membrane defects are advantageous to commercial practice.
Other methods for encapsulating or otherwise immobilizing biologically active materials, e.g. viable cells, have been disclosed which involve suspending the biologically active material in a gel composition and incorporating the gel material into the pores of a semi-permeable or permeable structure, or reacting the gel material to form a porous polymeric coating over the gel material. For example, U.S. Pat. No. 5,116,747 to Moo-Young et al. describes the immobilization of cells and other biologically active materials within a semipermeable membrane or microcapsule composed of an anionic polymer such as alginate induced to gel in the presence of calcium and/or a polymeric polycation such as chitosan.
U.S. Pat. No. 4,663,286 to Tsang et al. describes the encapsulation of solid core materials such as cells within a semipermeable membrane, by suspending the core material in a solution of a water-soluble polyanionic polymer, preferably alginate salts, forming droplets, and gelling the polyanion with a polyvalent polycation such as a polypeptide, a protein or a polyaminated polysaccharide, preferably polylysine, polyarginine, or polyornithine. This patent further teaches controlling the porosity and permeability of the disclosed compositions to molecules ranging from about 60,000 to about 900,000 Daltons by changing the degree of hydration of the polymer. Incubation in saline or chelating agents increases hydration and expands the gels, whereas incubation in calcium chloride contracts the gel mass. Increases in charge density of the polycationic membrane generally produces smaller pores. Increases in the molecular weight of the polycationic polymer generally produces a thicker, less permeable membrane.
U.S. Pat. No. 4,803,168 to Jarvis describes the encapsulation of core materials such as cells, enzymes, antibodies, hormones, etc. within a semipermeable membrane or microcapsule composed of an aminated polymeric inner layer such as chitosan ionically bound to an anionic polymeric outer layer such as polyglutamic or polyaspartic acid.
While these other methods may provide a generally useful means for encapsulating cells, techniques involving the formation of a membrane around a gel core material containing the cells can have certain drawbacks. As discussed above, it is difficult to fine tune the membrane porosity and to form defect free membranes. Moreover, there can be drawbacks in trying to store cells using such materials. Viable cells are typically stored by freezing, for example, in liquid nitrogen. However, the gel core materials and polymeric coating can become brittle and difficult to handle when inserted in a liquid nitrogen environment. Thus, it would be commercially advantageous to provide membranes having controlled porosity and physical integrity which are useful for containing viable cells and which exhibit excellent mechanical handling ability even when frozen, e.g. in liquid nitrogen. It would also be advantageous to provide a system for containing viable cells which can be formed around the cells under mild conditions without the need for a gel core material carrier for the cells.
Polymeric membranes produced by an electrospinning technique have been suggested as being useful for biological membranes such as substrates for immobilized enzymes and catalyst systems, wound dressing materials and artificial blood vessels, as well as for aerosol filters and ballistic garments.
Electrospinning is an atomization process of a conducting fluid that exploits the interactions between an electrostatic field and the conducting fluid. When an external electrostatic field is applied to a conducting fluid (e.g., a semi-dilute polymer solution or a polymer melt), a suspended conical droplet is formed, whereby the surface tension of the droplet is in equilibrium with the electric field. Electrostatic atomization occurs when the electrostatic field is strong enough to overcome the surface tension of the liquid. The liquid droplet then becomes unstable and a tiny jet is ejected from the surface of the droplet. As it reaches a grounded target, the material can be collected as an interconnected web containing relatively fine, i.e. small diameter, fibers. The resulting films (or membranes) from these small diameter fibers have very large surface area to volume ratios and small pore sizes. Although membranes can be produced by electrospinning under mild conditions, no practical industrial process has been implemented for producing membranes useful for medical applications. This is because with the production of small fibers, such as nanosize fibers, the total yield of the process is very low and a scale-up process, which maintains the performance characteristics of the films (or membranes), cannot be easily achieved.
Thus, there is a need for improved cell storage and delivery systems which can be produced on an industrial scale, which do not have the above-mentioned disadvantages.
According to the present invention, it has now been found that viable cells can be stored and later delivered to a mammal with a delivery system that contains a biodegradable and/or bioabsorbable matrix which avoids the above-mentioned disadvantages.
In one aspect, the invention relates to a cell delivery system which includes a biodegradable and/or bioabsorbable fibrous matrix and viable cells physically associated with the matrix as a carrier whereby the cells are contained and released at a controlled rate.
Preferably, the biodegradable and/or bioabsorbable fibrous matrix is formed by electrospinning fibers of biodegradable and/or bioabsorbable fiberizable material.
Preferably, the matrix will contain electrospun fibers having fiber diameters in the range from about 10 up to 1,000 nanometers, more preferably, in the range from about 20 to about 500 nanometers.
In one embodiment, the matrix contains a composite of different biodegradable and/or bioabsorbable fibers.
In another embodiment, the matrix contains an asymmetric composite of different biodegradable and/or bioabsorbable fibers.
Different fibers can include fibers of different diameters, fibers of different biodegradable and/or bioabsorbable materials, or fibers of both different diameters and different biodegradable and/or bioabsorbable materials.
Preferably, the matrix will contain at least about 20 weight percent of submicron diameter fibers, more preferably, at least about 50 weight percent of submicron diameter fibers.
The biodegradable and/or bioabsorbable fiberizable material is preferably a biodegradable and/or bioabsorbable polymer. The biodegradable and/or bioabsorbable polymer preferably contains a monomer selected from the group consisting of a glycolide, lactide, dioxanone, caprolactone, trimethylene carbonate, ethylene glycol and lysine.
In one embodiment, the biodegradable and/or bioabsorbable polymer is a biodegradable and/or bioabsorbable linear aliphatic polyester, preferably a polyglycolide or a copolymer poly(glycolide-co-lactide).
The biodegradable and/or bioabsorbable fiberizable material can also include a material derived from biological tissue, e.g., collagen, gelatin, polypeptides, proteins, hyaluronic acid and derivatives or synthetic biopolymers.
The fibers of different biodegradable and/or bioabsorbable materials can include fibers having different chemical composition, such as different polymeric materials, different molecular weights of the same polymeric material, different blends of polymers, materials having different additives or materials having different concentration of additives.
In another embodiment the matrix will contain different fibers, i.e. different diameters and/or different materials, having diameters in the range from a few nanometers up to almost about one micron, more preferably about 10 up to about 1000 nanometers and most preferably from about 20 to about 500 nanometers. The fibers of different diameters can include both fibers having diameters less than 300 nanometers and fibers having diameters greater than 300 nanometers.
In yet another embodiment, the fibrous matrix is formed by electrospinning different fibers of different materials, in which the article contains a composite of different fibers. Preferably, the composite of different fibers will contain submicron diameter fibers. The composite can be an asymmetric composite.
The matrix can also contain small blobs of biodegradable and/or bioabsorbable material. Preferably, the small blobs will have diameters in the range of about 20 to about 500 nanometers and, more preferably, about 200 to about 1500 nanometers.
In one embodiment, the article also contains at least one cell culture additive (as defined below). Examples of cell culture additives include additives which effect cell-membrane affinity, such as peptites, proteins, hyaluronic acid, as well as hydrophilic monomers, oligomers or polymers. The additive can be contained at the surface and/or within the biodegradable and/or bioabsorbable material itself, including within the fibers or within the small blobs of material, if present. In such a case, the fibers (and/or small blobs) can contain different concentrations of the additives or different additives.
The matrix can also have the structure of a plurality of layers, wherein at least one of the layers is a composite (or asymmetric composite) of different biodegradable and/or bioabsorbable fibers. In such a case, the article can also contain at least one additive between at least two of the layers.
Preferably, the cell delivery system is formed by electrospinning fibers of biodegradable and/or bioabsorbable fiberizable material in a layered structure, which includes:
a relatively thick base layer of a biodegradable and/or bioabsorbable fibrous matrix,
a dispersion of viable cells dispersed over the surface of the base layer,
a relatively thin top layer of a biodegradable and/or bioabsorbable fibrous matrix covering the dispersion of cells and adhered to the surface of the base layer, wherein the top layer has sufficient porosity to allow transfer of O2 and nutrients from outside the top layer to the cells and transfer of CO2 from the cells to the outside of the top layer, and
an optional porous partition layer to separate the sandwich layers.
The base layer preferably has a thickness in the range of about 20 to about 500 microns and, more preferably, about 50 to about 250 microns.
The top layer preferably has a thickness in the range of about 1 to about 50 microns and, more preferably, about 5 to about 20 microns.
The optional partition (or spacer) layer preferably comprises a biodegradable and/or bioabsorbable material having a relatively fast adsorption rate. Examples of such materials are the same as those made up of the bottom layer but with a different composition and morphology so as to have larger (bicontinuous) pores of sizes ranging from 1 to 5 microns and with a faster degradation rate in order to keep the embedded cells alive. The partition layer preferably has a thickness of about 1 to about 200 microns and, more preferably, about 5 to about 100 microns. It should have the ability for fast replacement by fluids that contain nutrients and oxygen for the cells and that can remove carbon dioxide and wastes. In one embodiment the thin top layer and the optional spacer layer can be integrated into one single layer that performs these functions.
The cell delivery system is preferably capable of being frozen and returned to room temperature without adversely affecting the integrity of the fibrous matrix (or layers) or the viability of the cells.
The cell delivery system can be used in connection with a variety of tissue precursor cells including differentiated cells obtained (e.g., harvested) from tissue of the lung, liver, kidney, thymus, thyroid, heart, brain, pancreas, bone, and the like. Tissue precursor cells can also include cells which are pre-selected to differentiate into specific cell types or so-called xe2x80x9cstemxe2x80x9d cells (or xe2x80x9cprogenitorxe2x80x9d cells) that are undifferentiated precursor cells.
The tissue precursor cells can include any of the following: epidermal cells, chondrocytes and other cells that form cartilage, macrophages, dermal cells, muscle cells, hair follicles, fibroblasts, organ cells, osteoblasts and other cells that form bone, endothelial cells, mucosal cells, pleural cells, ear canal cells, tympanic membrane cells, peritoneal cells, Schwann cells, corneal epithelial cells, gingiva cells, neural cells, neural stem cells such as central nervous system (CNS) stem cells, e.g., spinal cord or brain stem cells, as well as autonomic nervous system (ANS) stem cells, e.g., post-ganglionic stem cells from the small intestine, bladder, liver, lung, and heart, (for engineering sympathetic or parasympathetic nerves and ganglia), tracheal epithelial cells, hepatocytes, pancreatic cells, and cardiac cells. The tissue precursor cells can also be neuroendocrine stem cells.
In another aspect, the cell delivery system can be used with isolated, mammalian adult autonomic nervous system neural stem cells. These stem cells can be isolated from any innervated tissues in the body, including the heart, bladder, intestine, lung, liver, and kidney tissue. The invention also includes isolated, mammalian neuroendocrine stem cells, e.g., adult cells, such as those isolated from the pancreas (adult or fetal), or adult cells isolated from the adrenal medulla.
The cell delivery system can contain a supply of nutrients for the cells. Examples of nutrients include sugars, amino acids, growth factors, vitamins, hormones, cytokines, etc. The nutrients can be contained between the layers and/or within the fibers.
In another aspect, the invention is directed to a method for storage of living cells for delivery to a mammal. The method includes:
(a) providing a cell delivery system which contains:
(i) viable cells; and
(ii) a biodegradable and/or bioabsorbable fibrous matrix as a carrier physically associated with the cells to contain and release the cells at a controlled rate;
(b) cooling the cell delivery system down to a preservation temperature under conditions which maintain the integrity of the system; and
(c) maintaining the cell delivery system at or below the preservation temperature until a time when cell delivery is desired.
The preservation temperature is preferably below at least about xe2x88x9250xc2x0 C. and the cooling step is preferably carried out by submerging the cell delivery system in liquid nitrogen.
In yet another aspect, the invention is directed to a method for delivery of viable cells to a mammal. The method includes:
(a) providing a cell delivery system containing:
(i) viable cells; and
(ii) a biodegradable and/or bioabsorbable fibrous matrix as a carrier physically associated with the cells to contain and release the cells at a controlled rate; and
(b) positioning the cell delivery system at a desired location for cell delivery to the mammal.
The invention can also be used as a method for delivering viable cells in the treatment of defective tissue. For example, it can be used for treating defective nervous tissue by locating the physical boundaries of the defective tissue; removing the defective tissue to create a cavity and exposing healthy nervous tissue at the surfaces of the cavity; loading a neural stem cell composition into a cell delivery system in the general size and shape of the cavity, wherein the neural stem cells are selected to differentiate into the healthy nervous tissue; and implanting the delivery system into the cavity, thereby treating the defective nervous tissue. The defective nervous tissue can be CNS tissue, e.g., in the brain or spinal cord, ANS tissue, or neuroendocrine tissue. The neural stem cells can be isolated from the healthy nervous tissue. In this method, a spacer can be implanted into the cavity temporarily, and then replaced with the cell delivery system.
The present invention provides cell delivery and storage systems containing a biodegradable and/or bio absorbable fibrous matrix and having improved performance and handling characteristics, including improved performance in both cell storage and cell delivery.
Additional objects, advantages and novel features of the invention will be set forth in part in the description and examples which follow, and in part will become apparent to those skilled in the art upon examination of the following, or may be learned by practice of the invention. The objects and advantages of the invention may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the appended claims.